Analytical methods that combine the selectivity of enzymes with the sensitivity of amperometric detection are of interest to the diagnostic industry. The reduction of the nicotinamide co-enzymes (NAD and NADP) is particularly important because they are produced in reactions catalyzed by dehydrogenases. Dehydrogenase catalyzed reactions according to the equation: ##STR1## play an important role in biological cells and analytical reactions. Several hundred different dehydrogenases are known which selectively catalyze the conversion of different substrates into products. When the substrate, e.g. glucose, is oxidized, the enzymes NAD.sup.+ and/or NADP.sup.+ are reduced to NADH and NADPH respectively. These co-enzymes are a necessary element in the reaction due to their ability to act with the dehydrogenase enzyme to form an energy-transferring redox couple. The pyridine linked dehydrogenases transfer reversibly two reducing equivalents from the substrate to the oxidized form of the pyridine nucleotide; one of which appears in the reduced pyridine nucleotide as a hydrogen atom, and the other as an electron. The other hydrogen atom removed from the substrate appears as free H.sup.+ in the medium.
The co-enzymes NAD.sup.+ and NADP.sup.+ are expensive chemicals making their regeneration and reoxidation to their original state imperative if they are to be economically used in low cost, disposable, analytical devices.
NADH is oxidized directly at different base electrode materials only with high overvoltages on the order of 1 volt. However, a decrease in this overvoltage can be obtained by the adsorption of functionalities on the electrode surface which mediate the electron transfer from NADH to the electrode. Such mediators are typically selected from materials which may be reoxidized electrochemically without excessive overvoltages rendering them useful as an auxiliary system for electrochemical regeneration. Various mediator compounds suitable for this purpose are known. In U.S. Pat. No. 4,490,464 there are mentioned, by way of background, mediators such as phenazine methosulfate (PMS); phenazine ethosulphate (PES); thionine and 1,2-benzoquinone. This patent goes on to describe electrodes which are modified to catalyze the oxidation of NADH, NADPH or analogs thereof by imparting to the electrode surface as mediator a condensed aromatic ring system comprising at least three and preferably four or more condensed aromatic rings with or without heteroatoms. More particularly, this patent describes the electron exchange with the co-enzyme or analog thereof by structural elements comprising one of either alkyl phenazinium ions, phenazinium ions, phenazinones, phenoxazinium ions, phenoxazinones, phenothiazinium ions or phenothiazinones.
In J. Electroanal. Chem. 287, 61-80 (1990) there is disclosed 3-.beta.-naphthoyltoluidine blue O (I): ##STR2## which is perhaps the most effective of the known phenothiazinium mediators generically disclosed in the '464 patent. A variety of the mediators disclosed in this patent are compared in J. Electroanal. Chem. 292, 115-138 (1990).
The phenoxazinium and phenothiazinium ions disclosed in the '464 patent are positively charged species such as (I) above and are readily distinguishable from the mediator compounds of the present invention. The phenoxaziones and phenothiazinones claimed in the '464 patent are 3H-phenothiazines (II) and 3H-phenoxazines (III): ##STR3## in which the 3-position is derivatized with a carbonyl oxygen group. They bear a structural resemblance to the compounds of the present invention in that the oxygen atoms in (II) and (III) are replaced by nitrogen atom bearing substituted phenyl rings. In reality, however, these compounds are quite different and there is no suggestion in the prior art that replacing the carbonyl oxygen of compounds (II) and (III) with a phenyl-substituted nitrogen atom would afford effective mediators.
The compounds, whose utility as mediators is taught herein, are disclosed in U.S. Pat. No. 4,710,570 which describes the "leuko" or reduced form of these dyes to be "suitable as dye-forming agents in pressure sensitive, thermographic, photothermographic and photographic imaging systems."
U.S. Pat. No. 5,264,092 discloses the mediators of the '464 patent covalently attached to polymers which are useful for the electrochemical regeneration of NADH. This patent discloses a variety of polymeric backbones to which the mediators are attached. Polymer/mediator modified electrodes are also disclosed. Certain of the mediators of the present invention also perform well when immobilized on polymers.